Substrate deactivation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase by erythrose 4-phosphate

3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS, EC 4.1.2.15) catalyzes the condensation of phosphoenolpyruvate (PEP) with erythrose 4-ph osphate (E4P) to give DAH7P via an ordered sequential mechanism. In the abs ence of PEP (the first substrate to bind), E4P binds covalently to the phen ylalanine-sensitive DAH7PS of Escherichia coli, DAH7PS(Phe), deactivating t he enzyme. Activity is restored on addition of excess PEP but not if deacti vation was carried out in the presence of sodium cyanoborohydride. Electros pray mass spectrometry indicates that a single E4P is bound to the protein. These data are consistent with a slow, reversible Schiff base reaction of the aldehydic functionality of E4P with a buried lysine. Molecular modeling indicates that Lys186, a residue at the base of the substrate-binding cavi ty involved in hydrogen bonding with PEP, is well placed to react with E4P forming an imine linkage that is substantially protected from solvent water .